Membrane proteins such as G-Protein Coupled Receptor (GPCR), other receptor proteins, enzymes, transport proteins and ion channels or other transmembrane proteins or proteins anchored to the biological membrane play a major role in the functioning of a cell, especially in relation with its growth control, the regulation of physiological functions, signaling and mediation of cellular transfer [Almen, M. S et al.: “Mapping the human membrane proteome: a majority of the human membrane proteins can be classified according to function and evolutionary origin”; BMC Biology; 2009; 7; 50]. Membrane proteins are amphiphilic macromolecules having hydrophilic and hydrophobic regions which across one or several times the biological membrane. The hydrophobic regions of certain amino acids bear apolar side chains which are folded in a helix and b barrel forms. Each of these secondary structures representing a TransMembrane Domain (TMD) have the hydrophobic parts of their amino acid directed towards the outside of the a helix, and contract by weak non-covalent hydrophobic interactions with the aliphatic chains of the lipids constituting the biologic membrane of cell, bacteria or virus. The cohesion of membrane proteins is also provided by the charged polar heads of lipids which induce Coulombic forces with their hydrophilic loops localized in the extracellular regions [Eva Pebay-Peyroula: Biophysical Analysis of Membrane Proteins, Wiley-VCH Books, 2008].
It has been estimated that about 20 to 30% of the human genome codes for Integral Membrane Proteins (IMP) [J. Nilsson et al., Proteins: Struct., Funct., Genet., 2005, 60, 606] and more than 50% which are involved in signaling pathways in the most serious forms of human diseases such as cancer, Alzheimer's disease, diabetes or malaria, making them particularly promising targets for drug development [John P. Overington et al.: “How many drug targets are there?”; Nature Reviews Drug Discovery, 5, 993-996, 2006].
However, in spite of progress made during the past years, the quantity of three dimensionally resolved structures of membrane proteins only accounts for about 2% of the 92 000 structures that have been posted in the Protein Data Bank (PDB) in 2014.
Membrane proteins can include one or more subunits together with hydrophobic co-factors such as lipids, saccharides, peptides or proteins. For most biochemical and biophysical in vitro studies, membrane proteins need to be extracted from the lipid bilayer by using synthetic compounds such as detergents.
Detergents are amphiphilic compounds with a structure comprising both polar and apolar distinct domains which confer them a good solubility in water. Detergents have surfactant properties and are able to adsorb on interfaces.
Self-assembly of detergents is a well-defined phenomenon ruled by the hydrophobic effect [C. Tanford, <<The Hydrophobic Effect: Formation of Micelles and Biological Membranes>>, 1st Ed., John Wiley & Sons, Inc., New York, 1973]. At low concentrations, the presence in solution of non-polar groups disturbs the hydrogen bonds network between water molecules, which constrains them to organize themselves around apolar parts of the detergents and allows their solubilization.
To minimize this unfavorable disturbance of water molecules, the detergents are exchanged between the solution and the surface of the water where they form a compact film which decreases the surface tension of water. The liquid surface becomes saturated with detergent molecules from a threshold concentration called the Critical Micellar Concentration (CMC). The further addition of detergent molecules induces a spontaneous self-assembly and forms aggregate assemblies in solution called micelles which are defined by a size, a shape and an aggregation number.
The role of detergent-based micelles is to disturb the constitutive lipids of membrane cells stabilizing membrane proteins and solubilizes them in Protein/Detergent Complexes (PDC) while maintaining their conformational states to certain extend.
Monomers of detergent insert into the lipid bilayer until achieving its saturation, cause fragmentation and formation of mixed lipid/detergent micelles by action of detergent/detergent cooperative interactions. Sufficient amounts of detergent dissolve entirely lipids of the bilayer which results to a phase transition from a lamellar to a micellar phase comprising mixed lipid/detergent micelles and solubilized PDCs where their transmembrane domains interact with the hydrophobic tails of detergents [Le Maire et al.: “Interaction of membrane proteins and lipids with solubilizing detergents”, Biochimica Biophysica Acta (BBA)—Biomembranes, 2000, 1508, 86-111].
Solubilization of membrane proteins therefore requires the use of detergents able to extract them from biological membrane and able to maintain them in aqueous solution under a conformational form by reproducing an environment around the protein similar to biological membranes with aid of structuring detergents. This extraction process still presents a major challenge related to the difficulties at handling over an extended period of time membrane proteins that tend to form aggregates in aqueous solution [M. Caffrey, “Membrane protein crystallization”, J. Structural Biology, 2003, 142, 108-132]. During this process, the dissociating properties of detergents tend to disrupt essential protein/protein and protein/lipid interactions inside the quaternary structure of proteins which are essential to keep a specific folding responsible of their biological functions. Solubilization within the micelles of the different subunits for oligomeric proteins, a removing of lipid cofactors stabilizing or an intrusion of detergent tails onto hydrophobic transmembrane regions can cause an improper folding and disabling of the protein [J. U. Bowie, Curr. Opin. Struct. Biol., 2001, 11, 397-402; C. Breyton et al., J. Biol. Chem., 1997, 272, 21892-21900].
In this context, many commercially available detergents lead to the denaturation and the aggregation of the membrane protein, which is often irreversible [G. Privé: “Detergents for the stabilization and crystallization of membrane proteins”, Methods, 2007, 41, 388-397]. Although a wide range of detergents for membrane protein research are available, there is no such thing as a “universal detergent” suitable to all protein studies.
According to the nature of their polar heads, detergents are commonly classified as ionic, zwitterionic or neutral. The neutral class is often related as mild detergents which are less denaturing against membrane proteins.
Detergents used in membrane protein biochemistry to extract and purify membrane targets are mostly neutral detergents comprising glycoside groups in the polar part of the amphiphilic structure among the most commonly used include Dodecyl β-D-Maltopyranoside (DDM) and Octyl β-D-Glucoside (OG).
Working on this basis, the scientific community has developed a new kind of amphiphilic topologies with glycoside residues in the polar part different from the conventional linear head/tail detergents [Popot, J.-L., Annu. Rev Biochem. 2010, 79, 737-775] such as the branch-chained maltoside detergents [Qinghai Zhang, “Design, Synthesis, and Properties of Branch-Chained Maltoside Detergents for Stabilization and Crystallization of Integral Membrane Proteins: Human Connexin 26”, Langmuir, 2010, 26, 11, 8690-8696], nonionic amphipols [Bazzacco, P. et al., “Trapping and Stabilization of Integral Membrane Proteins by Hydrophobically Grafted Glucose-Based Telomers”, Biomacromolecules, 2009, 10, 3317-3326], fluorinated surfactants [Breyton, C. et al., “Micellar and biochemical properties of (hemi)fluorinated surfactants are controlled by the size of the polar head”, Biophys. J., 2009, 97, 1077-1086], amphiphilic tripods [Chae, P. S et. al., ChemBioChem, 2008, 9, 1706-9], steroidal facial amphiphiles [Lee, S. C. et al., “Steroid-based facial amphiphiles for stabilization and crystallization of membrane proteins”, Proc. Natl. Acad. Sci. U.S.A., 2013, doi:10.1073/pnas.1221442110], tandem facial amphiphiles [Chae, P S; et al., J Am. Chem. Soc. 2010, 132, 16750-16752], maltose neopentyl-glycol amphiphiles [Pil Seok Chae et al., “Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins”, Nature Methods, 2010, 7, 1003-1008], glucose-neopentyl glyco amphiphiles [Chae, P. S. et al. “Glucose-Neopentyl Glycol (GNG) amphiphiles for membrane protein study”, Chem. Commun., 2013, 49, 2287-2289], Chae's Glyco-Triton detergents [Chae, P. S. et al. <<Carbohydrate-containing Triton X-100 analogues for membrane protein solubilization and stabilization>>, Mol. BioSyst., 2013, 9, 626] and steroidal glyco-diosgenin amphiphiles [Chae, P. S. et al., “A New Class of Amphiphiles Bearing Rigid Hydrophobic Groups for Solubilization and Stabilization of Membrane Proteins”, Eur. J. Chem., 2012, 18, 9485-9490]. Recently, a novel class of anionic detergents based on a rigid calixarene structure has been developed which allows to extract, solubilize, stabilize and purify a wide range of membrane proteins while conserving their native three-dimensional structure and maintaining them in a functional active form [Matar-Merheb, R. et al.: “Structuring Detergents for Extracting and Stabilizing Functional Membrane Proteins”; Plos One; 2011, 6]. This technology is also disclosed in the patent application US 2011/0144314 A1.
Calixarenes are macrocyclic compounds composed of phenolic units connected by an ortho-ortho methylene bridge forming a hydrophobic cavity that is capable of forming inclusion complexes with a variety of molecules [C. D. Gutsche; “Calixarenes”; Accounts of Chemical Research; 1983; 16; 161-170]. Some calixarenes have already been characterized, in particular the tetramers, hexamers and octamers as well as several calixarenes having an odd number of rings in their molecule. Calixarenes have found several applications in industry and are applied for example in enzyme mimetics, non-linear optics, ion sensitive electrodes or sensors, and more particularly in selective extraction of membrane proteins. In the latter case, calixarenes have been described in the patent application US 2011/144 314 A1 and US 2012/123088 A1.
These molecules have a cyclic platform comprising of four phenol units onto which is grafted a hydrophobic part on the lower rim and hydrophilic parts on the upper rim. The hydrophobic part is composed of a single aliphatic chain varying in length from one to sixteen carbon atoms. The hydrophilic part consists of three carboxylate groups.
It is relatively well established that membrane proteins display a higher level of basic residues at the cytosol-membrane interface creating an enrichment of positive charges on the intracellular membrane interface [Von Heijne, G.: “Membrane protein structure prediction: Hydrophobicity analysis and the positive-inside rule”; J. Molecular Biology, 1992, 225, 487-494]. Moreover, membrane proteins have a higher aromatic residue content localized at the membrane-water interface on their a-helical transmembrane segments which plays an important role to the assembly folding and stability [Hong, H. et al., “Role of Aromatic Side Chains in the Folding and Thermodynamic Stability of Integral Membrane Proteins”; J. Am. Chem. Soc., 2007, 129, 8320-8327].
Hence, these calixarene detergents confer a better packing of the transmembrane domains of extracted/purified membrane proteins and closer to that imposed by lipids in a bilayer membrane environment in comparison to that obtained by conventional detergents. Due to their structural pattern and higher rigidity, these detergents significantly improve stabilization of membrane proteins at several levels: (a) by allowing an efficient covering of hydrophobic transmembrane domains with aliphatic chains; (b) by establishing electrostatic interactions at the intracellular cytosol interface between positively charged amino acids on membrane protein loops and negatively charged carboxylate groups of the detergents and thus generating a salt bridges network in close proximity around the protein, and (c) by establishing a strong adsorption on hydrophobic domains by means of π-stacking interactions between phenolic components of the calixarene core and aromatic amino acid residues located at the interfaces.
Calixarenic detergents have demonstrated their usefulness for extracting and stabilizing a range of both eukaryotic and prokaryote membrane proteins such as ABC transporters from Bacillus subtilis expressed in Escherichia Coli, GPCR Class I from yeast, Human Receptors from HEK cells and viral proteins [A. Jawhari. et al.: “Native and full length membrane protein isolation for Drug Discovery”; 12th annual Drug Discovery on Target Conference, Boston; September 2013]. Despite the effectiveness and potential of these calixarene compounds, some of the target membrane proteins were not fully active after solubilization and/or cannot be solubilized. That is why the present inventors wanted to design other compounds that can complement the previous ones for improving the solubilization rate and/or the functionality conservation of membrane proteins.